The present invention relates to the discovery, identification and characterization of a G protein coupled receptor, referred to herein as T1R3, which is expressed in taste receptor cells and associated with the perception of sweet taste. The invention encompasses T1R3 nucleotides, host cell expression systems, T1R3 proteins, fusion proteins, polypeptides and peptides, antibodies to the T1R3 protein, transgenic animals that express a T1R3 transgene, and recombinant “knock-out” animals that do not express T1R3.
Also within the scope of the present invention are transgenic cells that are heterozygous or homozygous for a nonfunctional T1R3 gene. The invention also encompasses methods of producing T1R3 transgenic animals and cells, particularly transgenic animals and cells that express a non-native T1R3 protein, and knock-out animals and cells.
The invention further relates to methods for identifying modulators of the T1R3-mediated taste response and the use of such modulators to either inhibit or promote the perception of sweetness. The modulators of T1R3 activity may be used as flavor enhancers in foods, beverages and pharmaceuticals.
The sense of taste plays a critical role in the life and nutritional status of humans and other organisms. Human taste perception may be categorized according to four well-known and widely accepted descriptors, sweet, bitter, salty and sour (corresponding to particular taste qualities or modalities), and two more controversial qualities: fat and amino acid taste. The ability to identify sweet tasting foodstuffs is particularly important as it provides vertebrates with a means to seek out needed carbohydrates with high nutritive value. The perception of bitter, on the other hand, is important for its protective value, enabling humans to avoid a plethora of potentially deadly plant alkaloids and other environmental toxins such as ergotamine, atropine and strychnine. During the past few years a number of molecular studies have identified components of bitter-responsive transduction cascades, such as α-gustducin (McLaughlin, S. K. et al., Nature, 357: 563-569 (1992); Wong, G. T. et al., Nature, 381: 796-800 (1996)), Gγ13 (Huang, L. et al., Nat. Neurosci., 2: 1055-1062 (1999)) and the T2R/TRB receptors (Adler, E. et al., Cell, 10: 693-702 (2000); Chandrashekar, J. et al., Cell, 100: 703-711 (2000); Matsunami, H. et al., Nature, 404: 601-604 (2000)).
Meanwhile, umami taste seems to be mediated by modified versions of metabotropic glutamate receptors (mGluRs) known as mGluR4 (Chaudhari and Roper, Ann. N Y Acad. Sci., 855: 398-406 (1998)) and by G-protein-coupled receptors (GPCRs) at the cell surface.
However, the components of sweet taste transduction have not been identified so definitively (Lindemann, B., Physiol. Rev., 76: 719-766 (1996); Gilbertson, T. A. et al., Curr. Opin. Neurobiol., 10: 519-527 (2000)), and the elusive sweet-responsive receptors have neither been cloned nor physically characterized.
Based on biochemical and electrophysiological studies of taste cells the following two models for sweet transduction have been proposed and are widely accepted (Lindemann, B., Physiol. Rev., 76: 719-766 (1996); Gilbertson, T. A. et al., Curr. Opin. Neurobiol., 10: 519-527 (2000)). First, a GPCR-Gs-cAMP pathway-sugars are thought to bind to and activate one or more G protein coupled receptors (GPCRs) linked to Gs; receptor-activated Gαs activates adenylyl cyclase (AC) to generate cAMP; cAMP activates protein kinase A which phosphorylates a basolateral K+ channel, leading to closure of the channel, depolarization of the taste cell, voltage-dependent Ca2+ influx and neurotransmitter release. Second, a GPCR-Gq/Gβγ-IP3 pathway-artificial sweeteners presumably bind to and activate one or more GPCRs coupled to PLCβ2 by either the α subunit of Gq or by Gβγ subunits; activated Gαq or released Gβγ activates PLCβ2 to generate inositol trisphosphate (IP3) and diacyl glycerol (DAG); IP3 and DAG elicit Ca2+ release from internal stores, leading to depolarization of the taste cell and neurotransmitter release. Progress in this field has been limited by the inability to clone sweet-responsive receptors.
Genetic studies in mice have identified two loci, sac (determines behavioral and electrophysiological responsiveness to saccharin, sucrose and other sweeteners) and dpa (determines responsiveness to D-phenylalanine), that provide major contributions to differences between sweet-sensitive and sweet-insensitive strains of mice (Fuller, J. L., J. Hered, 65: 33-36 (1974); Lush, I. E., Genet. Res., 53: 95-99 (1989); Capeless, C. G. and Whitney, G., Chem Senses, 20: 291-298 (1995); Lush, I. E. et al., Genet. Res., 66: 167-174 (1995)). Sac has been mapped to the distal end of mouse chromosome 4, and dpa mapped to the proximal portion of mouse chromosome 4 (Ninomiya, Y. et al., In Chemical Senses, vol. 3, Genetics of Perception and Communication (ed. C. J. Wysocki and M. R. Kare, New York: Marcel Dekker), pp. 267-278 (1991); Bachmanov, A. A. et al., Mammal Genome, 8: 545-548 (1977); Blizzard, D. A. et al., Chem Senses, 24: 373-385 (1999); Li, X. et al., Genome, 12: 13-16 (2001)). The orphan taste receptor T1R1 was tentatively mapped to the distal region of chromosome 4, hence, it was proposed as a candidate for sac (Hoon, M. A. et al., Cell, 96: 541-551 (1999)). However, detailed analysis of the recombination frequency between T1R1 and markers close to sac in F2 mice indicates that T1R1 is rather distant from sac (˜5 cM away according to genetic data of Li et al. (Li, X. et al., Genome, 12: 13-16 (2001)); and more than a million base pairs away from D18346, the marker closest to sac. Another orphan taste receptor, T1R2, also maps to mouse chromosome 4, however, it is even further away from D18346/sac than is T1R1.
To thoroughly understand the molecular mechanisms underlying taste sensation, it is important to identify each molecular component in the taste signal transduction pathways. The present invention relates to the cloning of a G protein coupled receptor, T1R3, that is believed to be involved in taste transduction and may be involved in the changes in taste cell responses associated with sweet taste perception.
The present invention also encompasses non-human transgenic animals that do not express functional T1R3 protein, particularly knock-out animals, and transgenic animals that express a non-native T1R3 protein. Also within the scope of the invention are cells that do not express functional T1R3 protein, particularly knock-out cells, and transgenic cells that express a non-native T1R3 protein. Experimental model systems based on these animals and cells can be used to further define the role of the T1R3 receptor and its responses to different types of tastants and taste modulators, furthering our understanding of the molecular biology and biochemistry of taste. Such model systems would also be useful for screening for novel tastants, enhancers of desirable flavors, and blockers of undesirable flavors.